Cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal HER2/neuexpressing tumors

Abstract

We have previously generated antihuman HER2/neuhumanized IgG3 fused to interleukin-2 (IL-2), IL-12, or granulocyte macrophage colony-stimulating factor (GM-CSF) [monofunctional fusion proteins (mono-AbFP)] or fused to IL-2 and IL-12 or IL-12 and GM-CSF [bifunctional fusion proteins (bi-AbFP)]. These AbFPs retained cytokine and antigen-binding activities. We have now further characterized the AbFPs and determined the heparinbinding activity of the fused cytokines, their ability to trigger IFN-; secretion and natural killer (NK) activation, and their direct antitumor efficacy. Flow cytometry revealed heparin-binding activity in the AbFPs containing IL-12 and IL-2, although this activity seems to be decreased in the bi-AbFPs. However, both bi-AbFPs retained the capacity to stimulate IL-12-dependent IFN-; secretion in the NK cell line KY-1, and IL-12/IL-2 bi-AbFP induced NK activity in splenocytes. The antitumor effectiveness of bi-AbFPs and mono-AbFP combinations was studied in mice challenged i.p. with three different human HER2/neu murine syngeneic models (D2F2/E2, CT26-HER2/neu, and MC38-HER2/neu). Although a significant variability in the profile of antitumor response was observed in the different tumor models, the combination of IL-12 and GM-CSF mono-AbFPs protected 100% of D2F2/E2-challenged and 75% of CT26-HER2/neu -challenged mice. In contrast, bi-AbFPs protected less than the combination of mono-AbFPs and, in some models, even less than monoAbFPs alone. However, in all cases, most of long-term survivors showed protection after s.c. rechallenge with the tumors and later with the parental tumors not expressing HER2/neu. These results show that, although the pattern of protection is tumor model dependent, treatments with AbFPs can effectively generate high levels of protection against peritoneal tumors expressing HER2/neu, which may be relevant in patients with primary or metastatic peritoneal carcinomatosis that may be observed in ovarian, colon, stomach, bladder, lung, and breast cancers.

Antibody–cytokine fusion proteins: applications in cancer therapy

“…This antibody-(IL-2) fusion protein retained the binding specificity of the antibody and cytokine biological activity similar to that of rhIL-2 [69]. We have also observed that anti-HER2/neu IgG3-(IL-2) retains the heparin-binding activity of IL-2 [70], a property that allows the binding of this cytokine to glycosaminoglycans present in the surface of cells [71] and that may play a role in the control of T-cell responses by IL-2 [72]. The authors explored the in vivo efficacy of anti-HER2 /neu IgG3-(IL-2) as a direct antitumor agent in a murine colon carcinoma cell line expressing human HER2/neu (CT26-HER2/neu), which was injected subcutaneously into immunocompetent syngeneic BALB/c mice.…”

“…Moreover, long-term survivors among mice challenged intraperitoneally with D2F2/E2 tumors retained significant protection after subcutaneous rechallenge with the same cell line more than 4 months later, and subsequent subcutaneous challenge with the parental cell line D2F2 not expressing human HER2/neu [70,124]. These results suggest that under these conditions, treatment with anti-HER2/neu (IL-12)-IgG3 could elicit a systemic long-term immune response and that this response could be expanded to antigens other than the targeted antigen [70,124]. In Section 5 the use of anti-HER2/neu (IL-12)-IgG3 in the context of prophylactic cancer vaccination is discussed.…”

“…In vitro assays demonstrated that this anti-HER2/neu (IL-12)-IgG3 (Figure 2A (II)) retained the capacity to induce proliferation of phytohemagglutinin (PHA)-activated PBMC in a dose-dependent manner similar to mscIL-12 alone [119]. In addition, since IL-12 is a heparin binding protein [121,122], we studied this activity in vitro and demonstrated that anti-HER2/neu (IL-12)-IgG3 retains the heparin-binding activity of IL-12 [70], including the ability to induce IFN-γ secretion from NK-susceptible lymphoma KY-1 cells [70]. The (IL-12)-antibody fusion protein also retained the ability to bind the human HER2/neu antigen expressed in the murine colon cancer cell line, CT26-HER2/neu [70].…”

“…In addition, since IL-12 is a heparin binding protein [121,122], we studied this activity in vitro and demonstrated that anti-HER2/neu (IL-12)-IgG3 retains the heparin-binding activity of IL-12 [70], including the ability to induce IFN-γ secretion from NK-susceptible lymphoma KY-1 cells [70]. The (IL-12)-antibody fusion protein also retained the ability to bind the human HER2/neu antigen expressed in the murine colon cancer cell line, CT26-HER2/neu [70]. In vivo studies in a direct therapeutic setting showed that anti-HER2/neu (IL-12)-IgG3 injected intravenously into immuno-competent mice challenged subcutaneously with CT26-HER2/neu cells significantly retarded tumor growth in comparison with the antibody alone [119].…”

“…In other studies, mice challenged intraperitoneally with epithelial syngeneic cancer models expressing human HER2/neu D2F2/E2 (mammary cancer cells, in a syngeneic BALB/c model), CT26-HER2/neu (colon cancer cells in a syngeneic BALB/c model), or MC38-HER2/neu (colon cancer cells in a syngeneic C57BL/6 model), and treated intraperitoneally with the anti-HER2/neu (IL-12)-antibody fusion protein, showed significant levels of protection in all tumor models [70]. Moreover, long-term survivors among mice challenged intraperitoneally with D2F2/E2 tumors retained significant protection after subcutaneous rechallenge with the same cell line more than 4 months later, and subsequent subcutaneous challenge with the parental cell line D2F2 not expressing human HER2/neu [70,124].…”